Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54

Biosorption of chromium by Termitomyces clypeatus

Sujoy K. Das 1 , Arun K. Guha ∗
Department of Biological Chemistry, Indian Association for the Cultivation of Science,
2A&B, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India
Received 14 May 2007; received in revised form 24 May 2007; accepted 25 May 2007
Available online 2 June 2007

Abstract
The manuscript describes removal of chromium from aqueous solution by biomass of different moulds and yeasts. The biomass of Termitomyces
clypeatus (TCB) is found to be the most effective of all the fungal species tested. The sorption of hexavalent chromium by live TCB depends on
the pH of the solution, the optimum pH value being 3.0. The process follows Langmuir isotherm (regression coefficient 0.998, χ2 -square 5.03)
model with uniform distribution over the surface which gets strong support from the X-ray elemental mapping of chromium adsorbed biomass.
The amino, carboxyl, hydroxyl, and phosphate groups of the biomass are involved in chemical interaction with the chromate ion forming a cage
like structure depicted by scanning electron microscopic (SEM) and Fourier transform infrared spectroscopic (FTIR) results. Desorption and FTIR
studies also exhibited that Cr6+ is reduced to trivalent chromium on binding to the cell surface. The level of chromium concentration present in the
effluent of tannery industries’ is reduced to a permissible limit using TCB as adsorbent.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Sorption; Termitomyces clypeatus; FESEM; FTIR; Chromium; Isotherm model

1. Introduction insoluble Cr(OH)3 at neutral pH, and becomes almost immobile

Chromium, a toxic heavy metal, dissipates into environ-
ment as a result of various industrial activities [1,2] such as
steel manufacturing, metal plating, mining, leather tanning, tex-
tile dying, cement industries etc. Of all the different oxidation
states trivalent and hexavalent chromium exist as stable species.
The hexavalent chromium species exists in aqueous solution
as oxyanionic entities like chromate (CrO4 −2 ), bichromate
(HCrO4 − ) and dichromate (Cr2 O7 −2 ), the relative distribution
of which depends on the solution pH [3,4]. Two other forms
of chromium, e.g. Cr3 O10 −2 , and Cr4 O13 −2 have also been
detected in highly acidic medium [4]. The oxyanionic entities
of Cr6+ do not bind to the negatively charged mineral surfaces,
e.g. silica or clay, become highly mobile in the environment
and soluble in a solution of neutral pH. In comparison, Cr3+
forms stable hydroxo complexes [e. g. Cr(OH)n (3−n)+ ] and the
cationic Cr3+ having strong affinity for particle surfaces yields
∗ Corresponding author. Tel.: +91 33 2473 4971/5904x502;
fax: +91 33 2473 2805.
E-mail addresses: sujoy iacs@yahoo.co.in (S.K. Das),
bcakg@mahendra.iacs.res.in, arunkumarguha@yahoo.com (A.K. Guha).
1 Tel.: +91 33 2473 4971/5904x502; fax: +91 33 2473 2805.
in the environment [5,6]. Cr3+ is also an essential micronutrient
compared with the toxic, mutagenic and carcinogenic hexava-
lent chromium [7] in addition to its reduced toxicity due to its
low bioavailability. In view of toxicity and related environmen-
tal hazards the level of chromium in wastewater must be reduced
to a permissible limit (5.0 mg/L and 0.5 mg/L for trivalent
and hexavalent chromium, respectively) [8] before discharg-
ing into water bodies. The removal of chromium employing
conventional methodologies [9,10] like ion exchange, chemi-
cal precipitation or reverse osmosis suffer from limitations like
high operating cost, incomplete precipitation, sludge genera-
tion, etc. On the other hand biosorption is receiving increasing
attention as an emerging technology for the removal of heavy
metals from contaminated effluents. The process is based on
the adsorption behavior of certain biological materials towards
organic or inorganic substances from their solution. Different
types of adsorbents [8,11–17] including activated carbon, saw-
dust, cactus leaves, lignin, spent grain, chitin, chitosan, jacobsite
(MnFe2 O4 ), etc. used for the removal of chromium results in low
removal requiring prolonged equilibrium time. Recently many
efforts have been directed towards the development of specific
biosorbents, the performance of which depends on the biomass
characteristics and microenvironment of target metal ion solu-

0927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2007.05.021
 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54
tion. The research focus for new biosorbents has in recent years
converged to microbial biomass [18–21] because the cell walls
of these types of biosorbents contain polysaccharides and pro-
teins having various functional groups such as amine, carboxyl,
hydroxyl, sulphates, and phosphates responsible for interacting
with the metal ions. Understanding the adsorption behavior is a
necessary prerequisite to the development of biosorption process
for industrial applications. This paper deals with our investiga-
tions on the sorption behavior of chromium on Termitomyces
clypeatus biomass, a mold strain available in large quantities as
a byproduct of enzyme and fermentation industries. The role of
different physicochemical parameters associated with the sorp-
tion of chromium from aqueous solution by T. clypeatus biomass
(TCB) through batch method is described. Information regard-
ing the binding sites and mechanism of sorption at molecular
level has been explored employing Fourier transform infrared
spectroscopy (FTIR), scanning electron microscopy (SEM), and
energy dispersive X-ray analysis (EDAX).
2. Materials and methods
2.1. Materials
Dehydrated microbiological media and ingredients were pro-
cured from Himedia, India. All other reagents were of analytical
grade and purchased from Merck, Germany and Sigma, USA.
2.2. Metal solution and analysis
A stock solution of chromium (1000 mg/L) was prepared
by dissolving potassium dichromate (K2 Cr2 O7 ) and chromic
chloride (CrCl3 , 6H2 O) separately in double distilled water and
diluted to get the desired concentration. Total chromium and
hexavalent chromium concentrations were measured by flame
atomic absorption spectrometer (Varian Spectra AA 55) and
UV–vis spectrophotometer (Varian model CARY 50 Bio) at
542 nm using diphenylcarbazide [22], respectively. The con-
centration of trivalent chromium was obtained by subtracting
the concentration of hexavalent chromium from that of the total
chromium.
2.3. Microorganisms
The fungi Rhizopus oryzae (MTCC 262), Mucor rouxii
(MTCC 386), Penicillium ochrochloron (MTCC 517),
Aspergillus viridie (MTCC 1782), and Pleurotus sajor-caju
(MTCC 141) were obtained from the Institute of Microbial
Technology, Chandigarh, India. T. clypeatus, Saccharomyces
cerevisiae MATa, Saccharomyces cerevisiae MATa,␣ and
Candida utilis were kindly supplied by Dr. A.K. Ghosh, Indian
Institute of Chemical Biology, Kolkata, India. The organisms
were maintained at regular intervals of 30 days as described in
our earlier publication [23].
2.4. Preparation of biosorbent
Live and dead biomass of yeast and mold were prepared as
described earlier [23] except that T. clypeatus was grown in
47
complex medium under static condition at 30 ◦ C for 7 days.
Growth medium contained 10% (v/v) potato extract and the
following ingredients in % (w/v): 1% malt extract, 0.057%
boric acid, 0.15% KH2 PO4 , 0.037% CaCl2 ·2H2 O, 0.05%
MgSO4 ·7H2 O, 0.0036% MnCl2 ·4H2 O, 0.03% ZnSO4 ·7H2 O,
0.025% FeSO4 ·7H2 O and 0.004% CuSO4 ·5H2 O.
2.5. Batch biosorption experiments
Biosorption experiments were conducted with 0.2 g
lyophilized mold or yeast biomass (live or dead) and 25 mL
of K2 Cr2 O7 solution containing 100 mg/L chromium taken in
100-mL Erlenmeyer flask, and incubated at 30 ◦ C (laboratory
temperature) for 48 h with shaking (130 rpm) unless otherwise
stated. At the end of incubation, biomass was separated by
centrifugation (10,000 rpm for 10 min) and the concentration
of chromium in the supernatant was determined by atomic
absorption spectrometer. The biomass which showed maximum
adsorption of chromium was selected for further studies. The
uptake of chromium by biomass was calculated using the fol-
lowing equation:
(C0 − Ce ) × V
qe =
W
where qe (mg/g) is the amount of chromium uptake by the
biomass, C0 (mg/L) and Ce (mg/L) the respective initial and
final metal ion concentrations in the solution, respectively, V
(L) the volume of the solution and W (g) is the weight of the
biomass.
The influence of hydrogen ion concentration on the sorption
of chromium was monitored at pH values 2.0–7.0 by suspending
0.2 g of dry live TC biomass in 25 mL 50 mM acetate buffer con-
taining either 100 mg/L Cr6+ or Cr3+ , other conditions remaining
the same.
The experiment for equilibrium sorption isotherm determi-
nation was conducted at pH 3.0 using 50 mM acetate buffer
as described above by varying Cr6+ concentration from 10 to
1000 mg/L.
2.6. Surface area measurement
The specific surface area of the lyophilized TCB was
determined by BET surface area analyzer (Quantachrome Instru-
ments, Autosorb-1-C). The dried biomass was initially degassed
by evacuation for 2 h at 80 ◦ C, and then the surface area was
determined by N2 sorption method [24].
2.7. Zeta potential
The Zeta potential of the particle was measured by deter-
mining the rate at which the particle moved in a known electric
field. This method is commonly known as microelectrophore-
sis. The surface charge characteristic of TCB at different pH
values was determined from Zeta potential measurement by
Zetasizer (Malvern Zetasizer) following the protocol of Li and
Bai [25].
48 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54

2.8. Scanning electron microscopy and energy dispersive
X-ray analysis
The samples of mold biomass for scanning electron micro-
scopic measurement were prepared according to the procedure
described by Smith et al. [26]. The micrographs were recorded
by FESEM (JEOL JSM-6700F) instrument equipped with
EDAX. Scanning electron micrographs were analyzed from
multiple test samples. X-ray elemental mapping was recorded
on FESEM instrument.
2.9. FTIR spectroscopy
threefold larger in comparison to yeast biomass. It is reported
[27,28] that the type of biomass has a significant effect on the
sorption process due to the variation in the cell size, morphology,
as well as the number of active binding sites and their distribu-
tion on the cell surface. Further, it was observed that the dead
biomass adsorbs 20–35% less chromium than the corresponding
live biomass under identical conditions. Reduction in chromium
uptake by the dead biomass is attributed to the loss of some
binding sites due to the high temperature required for inactiva-
tion of the biomass. Because of better uptake capacity towards
chromium, only live biomass of T. clypeatus was used to carry
out further studies in this respect.

Infrared spectra of pristine (protonated) and chromium 3.2. Characterization of the adsorbent surface
adsorbed TCB were recorded on a Nicolet-Magma 750 FTIR
spectrometer in the region 4000–400 cm−1 . The samples were The surface area of a solid is more important than the mass
pressed into spectroscopic quality KBr pellet with a sample/KBr for understanding and interpreting the ion sorption properties
ratio about 1/100. The FTIR spectra were recorded with 500 of the solid. The specific surface area of the lyophilized TCB
scans at a resolution of 2 cm−1 . determined by a BET surface area analyzer was 15.4 m2 /g. Low
specific surface area in comparison with activated carbon may be
due to either low surface porosity or smoothness of the surface
2.10. Removal of chromium from industrial effluents
[24].
The surface charge of the biomass as determined by mea-
Removal of chromium by T. clypeatus biomass was also
suring Zeta potential of its suspension at different pH values
tested with the actual effluent system. The effluents of tannery
varies from +10.2 mV to −34.6 mV with the corresponding
industries were collected in polypropylene container, from the
change of pH 2.0–7.0 (Fig. 1A). The net negative charge den-
Northern region of Kolkata, India, transported to the laboratory
sity increased corresponding change in the values of pH 3.5–7.0.
and stored at 4 ◦ C. The concentration of chromium and other ions
This observation suggests that the cell surface carries carboxy-
present in the effluents is summarized in Table 3. The concen-
late groups that have pK values within 3.5–5.0 [29] and the
tration of different ions was determined following the standard
phosphate groups with pK2 (the second dissociation constant of
methods. Removal of chromium was carried out at 30 ◦ C as
phosphoric acid) values within 7.0–8.0 [29]. Thus, the negative
described in batch biosorption experiments with 25 mL effluent
charge between pH 3.5–7.0 develops due to the carboxylate and
after adjusting its pH value to 3.0.
phosphate groups. The zero point charge (ZPC) is noted at pH
3.5. On the other hand, when the pH was higher than ZPC, a
3. Results and discussion net negative charge developed resulting from the deprotonation
of carboxylate and phosphate groups. The protonation of all the
3.1. Screening of microorganisms major functional groups e. g. carboxylic, phosphate and amino
resulted in a net positive charge at pH value less than the ZPC.
The biomass of different nonpathogenic molds and yeasts was
initially screened to study their uptake capacity of chromium 3.3. Effect of pH on chromium adsorption
from its solution. Live biomass of T. clypeatus was found to
be the most potent of all the biosorbents used (Table 1). It was The interaction between the metal ions and the functional
observed that the adsorption capacity of TCB was more than groups present on the biomass depends on the nature of the
biosorbent as well as the solution chemistry of the metal ions
Table 1 [30]. The pH of the solution greatly influences the metal specia-
Adsorption of chromium by different fungal biomass tion, sequestration and mobility [3,4,31,32]. In aqueous solution
Fungal strains Adsorption % chromium ions generally exist in two stable oxidation states,
Live Dead
trivalent and hexavalent. The former exists as Cr3+ , Cr(OH)+2
and Cr(OH)2 + in the pH range 1.0–6.0 and starts precipitating
Aspergillus viride (MTCC1782) 65 ± 4.0 42 ± 3.0 as Cr(OH)3 at a pH value > 6.0 [5,6], while Cr6+ forms H2 CrO4
± ±
and HCrO4 − species [3,4,32] at pH 2.0–3.0. Thus, at low pH
Mucor rouxii (MTCC 386) 70 3.0 55 2.0
Penicilium ochrochloron (MTCC 517) 75 ± 2.0 58 ± 3.0
Pleurotus sajor-caju (MTCC 141) 65 ± 3.5 50 ± 1.0 values oxyanionic species of Cr6+ is likely to be attracted by
Rhizopus oryzae (MTCC 262) 81 ± 4.5 61 ± 2.1 the positively charged functional groups present on the cell sur-
Termitomyces clypeatus 91 ± 2.1 62 ± 2.0 face, while opposite result would be expected for Cr3+ due
Candida utilis 32 ± 4.2 25 ± 1.1 to coloumbic interaction. It was observed that adsorption of
Saccharomyces cereviciae MATa 30 ± 3.1 22 ± 2.0
S. cereviciae MATa,␣ 35 ± 2.4 20 ± 2.2
Cr6+ increased slightly by increasing pH from 2.0 to 3.0 and
thereafter, decreased with further increase in pH values of the
 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54 49

Fig. 1. Effect of pH on chromium adsorption by Termitomyces clypeatus biomass at 30 ◦ C (A), (䊉) Cr6+ uptake, () Cr3+ , () Zeta potential; adsorption isotherm
of Cr6+ on T. clypeatus biomass (B), (䊉) experimental value, () non-linear Langmuir model, () non-linear Freundlich model; adsorption isotherm following
the linear form of Langmuir model (C); adsorption isotherm following the linear form of Freundlich model (D). Data represent an average of four independent
experiments ± S.D. shown by error bar.

solution, while maximum adsorption of Cr3+ took place at pH optimum pH of chromate reductase is around pH 7.0 and activ-
value 6.0 (Fig. 1A). Precipitation of Cr3+ as Cr(OH)3 at pH ity decreases significantly at low or high pH values [37]. So,
value > 6.0 restricted the experiment for solution having pH val- enzymatic reduction of Cr6+ to Cr3+ can be ruled out at low
ues beyond 6.0. Zeta potential measurements indicate that the pH. Accordingly we have measured the amount of both hexava-
overall surface charge remained positive at low pH values, which lent and trivalent chromium in the solution after adsorption with
facilitated the attraction of negatively charged oxyanionic chro- TCB at pH 2.0, 3.0, and 4.0. The results presented in Table 2
mate ions. On the other hand, decreased adsorption of Cr3+ at show the presence of both hexavalent and trivalent chromium
pH 2.0–3.0 is attributed to the competition of the binding sites in the aqueous solution after sorption of chromate with TCB.
for H+ ions. From the experimental data, it is noted that at pH This indicates that chromate being a strong oxidizing agent oxi-
2.0 adsorption of Cr6+ was less than 20% compared with that dizes some functional groups of biomass and in the process gets
at pH 3.0. Similar behavior of hexavalent chromium adsorption reduced to trivalent state. It was also observed that proportion
on a variety of biosorbents including fungal biomasses was also of trivalent chromium to hexavalent chromium decreased with
reported earlier [33–35]. However, the rationale behind such
influence of pH on the sorption process has not yet been ade-
Table 2
quately discussed. A number of factors are to be considered to Influence of pH on reduction of Cr6+
understand the role of pH in the sorption behavior of chromium
pH of the Uptake Total Cr Cr6+ mg/L Cr3+ mg/L Cr3+ /Cr6+
on TCB. Hexavalent chromium can be reduced to trivalent state
solution (mg/g) (mg/L)
both chemically [36] or enzymatically [37]. The cell wall of TCB
contains different electron donors and their close proximity to 2 8.77 25.69 3.25 22.44 6.9
3 11.15 7.25 1.51 5.74 3.8
aqueous chromate ions result in the formation of Cr3+ species.
4 7.03 39.61 24.36 15.25 0.63
This reduction is facilitated at low pH values. On the other hand,
50 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54
increasing in pH of the solution. Additionally, to confirm the
presence of both the forms of chromium on the biomass, we have
desorbed the chromium from the loaded biomass with 0.5 M
HCl or 0.5 M NaOH and measured the amount of both forms of
chromium in the eluent. The results show (data not shown) the
presence of both trivalent and hexavalent chromium but the per-
centage of Cr3+ being much higher than that of Cr6+ in the HCl
eluent while in NaOH eluent opposite results were observed.
This further confirms our hypothesis that chromate ions after
initial binding to the cell surface were reduced to Cr3+ . Thus,
the optimum pH value (3.0) can be explained as; at low pH
value positively charged functional groups adsorbed chromate
ions through anion exchange mechanism, however, as soon as
the pH was lowered below the optimum pH value, the chromate
oxidize the biomass and produced Cr3+ ions [36,38]. These Cr3+
ions then competed for the binding sites with the protons via the
cation exchange reaction resulting in low adsorption. The des-
orption of Cr3+ from the biomass at that low pH value also lead
to low uptake of chromium [38]. Consequently, the sorption of
Cr6+ from its aqueous solution by TCB was maximized at a pH
value which was high enough for anion exchange as well as
redox reaction to proceed simultaneously. Thus, we presumed
that the influence of hydrogen ion concentration on the present
sorption process is complex in nature and the noted optimum
pH value is the resultant of all these factors. However, 25% of
total adsorption also took place at a higher pH (6.0–7.0) value,
although cell surface contained negative charges. Based on this
experimental data we conclude that in addition to electrostatic
forces of attraction, other factors such as reduction, precipitation,
chemical interaction and physical forces such as hydrogen bond-
ing and or ion–dipole interaction also involved in the sorption
process.
3.4. Sorption isotherm
Sorption isotherm is a prerequisite to describe the stoi-
chiometric solute–solid interaction. A few parameters, such
as maximum sorption capacity, are important for optimizing
the design of sorption system, and analyzed using linearized
forms of the isotherm models of Langmuir [39] and Glas-
tone [40]. The result shows that the present sorption process
of chromium on the live TCB followed the Langmuir model
(Fig. 1, panels B and C) indicating chemisorption and monolayer
coverage of sorbate on the sorbent. The theoretical monolayer
saturation capacity (Qmax ) of the sorbate on the sorbent cal-
culated (54.05 mg/g) from the slope of the linearized curve of
Langmuir model (Fig. 1C) was very close to that obtained exper-
imentally from isotherm study (53.95 mg/g) (Fig. 1B), which
is higher than those reported for other types of biosorbents
[12–14,16,41]. Sorption capacity of live TCB towards chromium
was found to be higher than that reported for activated carbon
(15.47 mg/g) [18], though it covers much higher BET surface
area (500–3000 m2 /g) [42] compared to that of TCB (15.4 m2 /g).
Therefore, we summarized that besides BET surface area other
properties of the sorbent such as functional groups of the biomass
played important roles in the studied sorption process. The sorp-
tion phenomenon did not fit well with the Freundlich isotherm
model (Fig. 1, panels B and D) exhibiting deviation from linear-
ity over the entire concentration range.
3.5. Model analysis
Linear coefficients of determination, r2 , and non-linear χ2 -
square test, χ2 may be used to evaluate the ‘goodness of fit’ of
curves to the data they summarize and may be used to estimate
the probability of obtaining any series of deviations of observed
values from predicted values [43]. The value of linear coefficient
of determination, r2 , represents the percentage of variability in
the dependent variable that has been explained by the regression
line and may vary from 0 to 1. If there is no relationship between
the predicted values and actual values, the coefficient of deter-
mination is zero or very low, a perfect fit gives a coefficient of
1.0. On the other hand χ2 -square test, χ2 is basically the sum
of the squares of the differences between the experimental data
and data obtained from the models, with each square difference
divided by the corresponding data obtained by calculation from
the models. This can be represented mathematically as
 (qe − qe,m )2
χ2 =
qe,m
where qe,m is equilibrium uptake obtained by calculation from
the model (mg/g) and qe is the experimental data of the equi-
librium uptake (mg/g). χ2 -square, χ2 will be small number if
the experimental data are close to that obtained from model
and will be bigger if they differ. Therefore, it is necessary to
analyze the data sets using both linear coefficient of determi-
nation, and non-linear χ2 -square test to establish the best fit
isotherm model for the adsorption system. The coefficient of
determination, r2 , and Chi square test, χ2 , were determined
in the range of the whole metal ion concentration. The linear
regression coefficient, r2 , values were 0.998 and 0.965, respec-
tively for Langmuir and Freundlich models. On the other hand
the χ2 -square, χ2 , values of 5.03 and 18.53 for Langmuir and
Freundlich, isotherm, respectively with 9 degrees of freedom
(φ) corresponds to the significance level between 75–90% and
2.5–5% according Fischer and Yates chart [44]. Thus, Lang-
muir isotherm model appears to be the best fit for the present
adsorption process.
X-ray elemental mapping of the biomass after chromium
sorption (Fig. 2) depicts a uniform distribution of chromium
over the entire surface area which further supports the observed
Langmurian behavior of sorbate on the sorbent surface.
3.6. Chemical characterization of metal ion adsorption
In living cells the sorption mechanisms include both
metabolism dependent and independent processes. Metabolism
independent uptake process essentially involves cell surface
binding through ionic and chemical interaction, while dependent
process deals with the binding of both the surfaces followed by
intracellular accumulation [21,45]. The cell wall of the fungal
biomass generally contains large amounts of chitin, chitosan,
glucan and mannan as well as small amounts of glycoprotein
 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54 51

Fig. 2. X-ray elemental mapping of the biomass after chromium adsorption; carbon, nitrogen, oxygen, phosphorous, sulphur and chromium indicate the corresponding
element distribution on the biomass. Scale bar 100 ␮m.

[46,47]. These polymers are abundant sources of metal binding
ligands like carboxyl, amine, hydroxyl and phosphate groups.
Scanning electron microscopy along with energy dispersive
X-ray analysis has been used as a tool for biosorbent charac-
terization and elucidation of the probable mechanism involved
in the sorption process. Electron micrographs of the pristine
(protonated) (Fig. 3, panel A) and Cr6+ adsorbed (Fig. 3, panel
B) biomass clearly distinguish two cases. The surface of TCB is
rough and irregular with large area for metal-surface interaction
which formed a cage like structure after chromium sorption. The
exact reason for this formation is not known at this moment;
however, the chemical interaction between functional groups
of the biomass and chromate ion may be responsible for the
formation of cage like structures. Energy dispersive X-ray anal-

Fig. 3. Scanning electron micrographs of (A) pristine (protonated) biomass, (B) chromium adsorbed biomass; EDAX spectra of (C) pristine (protonated) biomass
and (D) chromium adsorbed biomass.
52 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54

ysis (EDAX) provides elemental information through analysis
of X-ray emissions caused by a high-energy electron beam. The
spectra recorded in spot profile mode, indicate the presence of C,
N, O, Na, P and S (Fig. 3, panel C). These signals are due to X-
ray emissions from the polysaccharides and proteins present on
the cell wall of the biomass. Additional signals of Cr are noted,
indicating the binding of metal ions on the biomass (Fig. 3, panel
D).
In order to understand the surface binding mechanism, it
is essential to identify the functional groups present on the
biomass involved in this process. The main effective binding
sites can be identified by FTIR spectral comparison of the pro-
tonated and Cr6+ adsorbed TCB after 24 h and 48 h incubation.
FTIR spectrum (Fig. 4A, curve 1) of the protonated biomass
exhibit the presence of amino, carboxyl, hydroxyl, phosphate
and sulphonate groups [48–51] on the unadsorbed biomass.
The strong bands in the region 3500–3000 cm−1 are the char-
acteristics of N H and O H stretching vibrations and those
around 2920–2850 cm−1 of alkyl chains. A distinct peak at
1745.5 cm−1 can be attributed to the C O stretching band from
the protonated carboxylic groups or ester groups, 1708.1 cm−1
for C O of the carboxylic groups of amino acids. The amide
I band is primarily a C O stretching mode and is centered at
1643.2 cm−1 . The amide II band is a combination of N H bend-
ing and C N stretching close to1550.0 cm−1 . Peak positions at
1541.0 cm−1 and 1413.7 cm−1 can be attributed to the COO− of
the carboxylate group of the biomass. The more complex amide
III band is located near 1330.0 cm−1 . The strong band within
1100–1000 cm−1 is due to C O bond, which is the characteris-
tic peak for polysaccharides. The absorption peaks in the region
of 1150.0 cm−1 , 1050.0–970.0 cm−1 and 1040.0–910.0 cm−1
for P O, P O C and P OH stretching, respectively, depict
the presence of phosphate groups in the biomass. The charac-
teristic band region at 800–850 cm−1 suggests the presence of
sulphonate group in the biomass.
Conspicuous changes (appearance or disappearance) in
the FTIR spectrum (Fig. 4A, curve 2) are observed after
chromium adsorption for 24 h on the biomass, the trans-

Fig. 4. (A) FTIR spectra of (curve 1) pristine (protonated) and (curve 2) chromium adsorbed biomass after 24 h of incubation, (B) chromium adsorbed biomass after
48 h of incubation,and (C) a schematic diagram of chromate–biomass interaction; Cr6+ initially binds with the functional groups of the biomass and then reduced to
trivalent chromium.
 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54
Table 3
Characteristics of effluents collected from tannery industries
Wastewater pH Temperature (◦ C) Concentration of Hg (mg/L)
A 5.0 31 5.82
B 6.0 30 10.54
C 4.5 29 20.85
D 6.8 30 11.92
a Chemical oxygen demand.
b Dissolved oxygen.
mittance at 3008.7 cm−1 disappeared and the bands at
1502.4 cm−1 , 1519.8 cm−1 , 1537.2 cm−1 and 1548.7 cm−1
shifted to 1552.6 cm−1 on adsorption. The observed changes
in the transmittance values in the range of 1450.4–1454.8 cm−1
and 1035.6–1043.4 cm−1 are closely related to N H bending
and C N stretching frequencies, respectively. Further, the peak
present at 1643.2 cm−1 shifted to 1631.7 cm−1 on chromium
adsorption. The shifting of these bands indicate the involvement
of N H of amines and C O of amides in the adsorption process.
Obvious the shifts of the carboxyl stretching band (1745.5 cm−1 )
to higher frequencies (1749.4 cm−1 ) and disappearance of peak
at 1708.1 cm−1 are observed in the chromium loaded biomass.
The shifting of absorption peaks at 1038.6–1043.5 cm−1 indi-
cates phosphate bond intervention in chromium adsorption. At
low pH value (3.0) carboxyl and phosphate groups were proto-
nated, thus suggesting the electrostatic interaction of carboxyl
and phosphate groups with chromium in the present adsorp-
tion process. The biomass after adsorption of chromium for
24 h (Fig. 4A, curve 2) shows the characteristics band of Cr6+ .
The characteristic vibrations bands of Cr6+ at 952.7 cm−1 ,
940.2 cm−1 , 920.4 cm−1 , and 750.1 cm−1 are observed in the
chromium adsorbed biomass. The formation of hydrogen bond
between the amino group and chromate ion ( NH· · ·O–CrO3 − )
is indicated by the appearance of band at 895.6 and 775.1 cm−1
[52]. However, after adsorption of chromium for 48 h, IR spec-
trum shows small changes in the region of 1000–500 cm−1
(Fig. 4B) as against after 24 h adsorption (Fig. 4A, curve 2).
The vibration bands for Cr6+ are noted, but their frequencies are
shifted and in addition, a new band at 528.2 cm−1 characteris-
tic of Cr(OH)3 also appeared (Fig. 4B). The shifting of Cr6+
band frequencies and the appearance of Cr(OH)3 band indicate
that Cr6+ initially binds to the functional groups of the biomass
and is then reduced to form Cr(OH)3 . FTIR study thus showed
that different functional groups of the biomass are involved
in chemical reactions [51,52], e.g. ionic interaction, hydrogen
bonding or ion–dipole interaction and complexation with chro-
mate ions. The cage like structures observed in SEM micrograph
on chromium adsorption may be due to the formation of both
intra and intermolecular complexes [53]. A schematic diagram
of chromate-biomass interaction is presented in Fig. 4C. The
scheme is based on desorption experiment, FTIR study and a
report [54] on the fluorescence detection of non-metal ions using
complexes between phosphate and tris(3-aminopropyl)amine.
We believe that the obtained results shed a new light on the
initial phase of interaction between Cr6+ and the living cells.
53
Concentration of other anions and cations (mg/L) CODa (mg/L) DOb (mg/L)
Ca+2 Na+1 SO4 −2 Cl−1
2.1 5.8 5.6 45 – 5.6
– 120.1 50.2 25 1.5 6.2
3.5 20.2 30.6 100 1.3 6.5
1.5 8.3 10.4 150 – 4.5
3.7. TCB–Cr interaction of industrial effluent
In order to explore the feasibility of TCB for treating
chromium, industrial effluent was used as feed solution. A
detailed composition of the wastewater samples obtained from
tannery industries in Kolkata, India is presented in Table 3. The
chromium concentration varied from 4.92 mg/L to 20.92 mg/L.
The pH values of the effluents were 5.0–7.0, depending on the
sites of collection. Therefore, the pH values of the wastewater
were adjusted to pH 3.0 prior to removal by TCB. The concen-
tration of chromium was brought down to 0.1 mg/L, 0.15 mg/L,
0.32 mg/L and 0.5 mg/L, respectively, after treatment with TCB.
This value is within the safe limit for discharging into water bod-
ies [8]. Presence of co-anions and cations has no inhibitory effect
at such low metal concentration in the present adsorption sys-
tem. The present study reveals the importance of TCB in the
effluent treatment containing chromium.
4. Conclusions
The present study has revealed new insight into the initial
cell surface binding of chromium on live biomass of T. clypea-
tus. The sorption process was found to depend on the pH of the
solution, pH 3.0 being the optimum value. The process follows
the Langmuirian isotherm model exhibiting uniform distribu-
tion of chromium over the surface of TCB. The results of FTIR
study demonstrate the interaction of functional groups of the
biomass with the chromium. The adsorbed chromate is reduced
to trivalent form which finds support from the FTIR and desorp-
tion studies on the chromium loaded biomass. Scanning electron
microscopic studies depict the formation of cage like structures
on sorption of chromium, while X-ray elemental mapping of
the chromium loaded biomass shows uniform distribution of the
metal ion over the mycelial surface. It is also observed that TCB
decreases chromium concentration of tannery industries’ efflu-
ent to a permissible limit. The present study offers a scope for
significant technological development towards the removal of
chromium from wastewater.
Acknowledgement
We thank Mr. S. Dey (Indian Institute of Chemical Biol-
ogy, Kolkata) for his cooperation during Electron Microscopic
analysis. Thanks are due to Ms. Mousumi Basu (Institute of
Environmental Studies and Wetland Management, Kolkata)
54 S.K. Das, A.K. Guha / Colloids and Surfaces B: Biointerfaces 60 (2007) 46–54

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